Frederick R. Maxfield - US grants

Receptor-mediated endocytosis is an important step in many biological processes including regulation of surface receptors, uptake of iron and cholesterol, entry of some viruses and toxins, clearance of proteases form extracellular fluids, and antigen presentation. Molecules or particles brought into the cell by endocytosis can reach various cellular destinations. Many receptors recycle back to the cell surface as their ligands are degraded. Viruses and toxins enter the cytosol. A key step in many of the sorting events is exposure to a low pH in an endosome. There are several distinct types of endosomes. These include early peripheral small visicles, a sorting endosome (CURL), and two post-segregation endosomes: one returning to the cell surface and one leading to lysosomal degradation. Recent work from this laboratory has shown that the different types of endosomes maintain different pH vlaues. It is proposed that changes in acidity in part determine the functional properties of different types of endosomes. Using fluorescein labeled molecules as a pH proble, the pH of each of the specific types of endosomes will be measured. Most important will be the pH in the sorting endosome, which has never been measured apart from other types of endosomes. Mechanisms of pH regulation will also be studied in living cells by measuring the pH of specific compartments under altered contitions (temperature, drugs, ionic environment, etc). The origins and fates of these various endosomes and their interrelationship is not known. The fusions and separations of these endosomal compartments with each other will be studied using fluorescence microscopy, biochemical techniques, and electron microscopy. This should establish the lifetime of the sorting endosome and indicate whether the recycling endosomes can serve as sorting organelles. The molecular signals for carrying receptor proteins on the recycling pathway or to lysosomes will be investigated using transfectants of normal or altered EGF and transferrin receptors.

We have developed an accurate and practical method for measuring cytoplasmic [Ca2+] in the light microscoope. The method involves the use of the calcium-sensitive fluorescent dye Quin 2. The ratio of fluorescence intensities with excitation at 340 nm and 360 nm (I340/I360) provides a measure of [Ca2+] which is independent Quin 2 concentration. Using image intensification fluorescence microscopy and digital analysis of video images we are able to measure I340/I360 (and cytoplasmic [Ca2+]) throughout the cell. The video image analysis system provides enough temporal resolution, spatial resolution, and sensitivity to detect [Ca2+] gradients within a single cell using Quin 2. First, we will refine methods for measurement of localized [Ca2+]. We will then use the method to determine whether significant [Ca2+] gradients are developed in various cell types under a variety of physiological conditions. We have already found that mitotic cells have a considerable local variation in cytoplasmic [Ca2+]. Cell types to be studied including macrophages during phagocytosis, rat basophilic leukemia cells with clustered IgE receptors, and neutrophils exposed to chemotactic peptide. We will examine the contributions of ion pumping, diffusion, and buffering in dissipating [Ca2+] gradients, and we will try to obtain data on the sites of Ca2+ release into the cytoplasm. The role of intracellular messengers for the release of Ca2+ will be studied by micro-injecting putative messengers into the cytoplasm of Quin 2-loaded cells and observing changes in I340/I360. These studies will complement a large number of studies from several laboratories which are providing evidence for overall changes in cytoplasmic [Ca2+] throughout the cells. Local [Ca2+] gradients may be important for localized cell responses to external stimuli or for local control of the cytoskeleton.

This is a request for a very flexible, modular system for optical microscopy of cells and tissues. The overall system is designed to take advantage of several recent developments in optical microscopy techniques, and it will allow integration of future methods as they become available. The system incorporates several types of optical microscopes. It includes distributed computer control of microscopes and analysis of data linked via high speed network to a larger centralized image processing facility. The image analysis facility will have a powerful image processing computer, large disks and tape systems for digital storage of image data, monitors for viewing processed images, and hardcopy devices for producing photographs of video images. In addition to analyzing images obtained with the two requested microscopes, the image analysis computer can be used for analyzing and storing images obtained on other microscopes in the users' laboratories. Most important, the components have been chosen for ease of use without committing large amounts of time to becoming familiar with the individual techniques. Overall, the system will make the most modern optical microscopy techniques available to all members of the user group and to others at the College. The microscopes requested will be used for scanning confocal fluorescence microscopy, video enhanced Nomarski microscopy, microscope spectrofluorometry, and image intensification fluorescence microscopy. The users' applications span a wide range of biological problems, including developmental biology, immunology, cell motility, mitosis, receptor- mediated endocytosis, renal physiology, and neuroanatomy among others.

The goal of this work is to understand (1) the mechanisms of intracellular calcium regulation in motile cells, (2) the relationship between calcium changes and other intracellular signalling mechanisms, and (3) the role of these signalling mechanisms in regulating cell motility. The proposed work will examine two types of phagocytic cells: the human neutrophil and the mouse peritoneal macrophage. For both types of cells, there is extensive biochemical characterization of the signal generating mechanisms and the mechanisms for regulation of the cytoskeleton. There is also substantial information on the motile behavior of these cells in response to various stimuli. Despite intensive study, the mechanisms for controlling motility in vivo remain unclear, although roles for (Ca2+)i, pHi, kinase C, and other signals have been proposed and investigated. Optical microscopy techniques will be used to measure both the generation of intracellular signals and the motile response in single cells. (Ca2+)i will be measured with high temporal and spatial resolution. These measurements will be made simultaneous with measurements of pHi and cell morphology to provide detailed correlations between the generation of signals and the motion of cells. Methods to measure kinase C activation in single cells will be developed. The interplay between three signals ((Ca2+)i, pHi, and kinase C) will be examined. Both chemotaxis and phagocytosis exhibit desensitization/resensitization phenomena. The mechanism for this will be studied based on analysis of the properties of these three intracellular signals and their inter-relationships. Finally, the role of these signals in the morphological and cytoskeletal changes will be examined. Both chemotaxis and phagocytosis are processes that require study at the single cell level to understand how biochemical mechanisms are orchestrated to produce specific cell behavior. The microscopic methods described in this proposal provide a means to study intracellular signals and cell responses at the single cell level.

The goal of this project is to understand the endocytic mechanisms for routing ligands and receptors to different destinations and to determine how this routing is a determinant of physiological consequences. Receptor-mediated endocytosis is important in many biological processes, including uptake of LDL and delivery to lysosomes, internalization of transferrin and release of iron in acidified endosomes, clearance of signalling ligands such as insulin or EGF from the surface of cells, antigen presentation, infection by many types of viruses, and entry of certain toxins into cells. There is evidence that endocytosis and recycling is the mechanism for insulin-regulated redistribution of hexose transporters, -and it has been suggested that the defect in cystic fibrosis may alter endocytic trafficking. Mechanisms for targeting ligands and receptors after endocytosis are not clear. Even the most basic relationships among the organelles involved are not understood and agreed upon by researchers in the field. In this project, optical microscopy will be used to directly observe and measure endocytic processes. There have been dramatic increases in the power of microscopy for studying living cells which provide a unique opportunity to observe the trafficking of nearly native molecules in minimally perturbed intact cells. Bright, photostable fluorescent derivatives of natural ligands such as LDL and transferrin will be used to directly observe dynamic interactions among endocytic organelles using confocal microscopy and image intensification fluorescence microscopy. Digital image analysis will be used to quantify the kinetics of ligands moving into and out of individual,identified endocytic organelles. Properties of the organelles such as their lifetimes and rate of fusion with other endosomes will be quantified. In addition to studying natural ligands and receptors, the behavior of fluorescent lipids, GPI-anchored proteins, modified receptor proteins, and multivalent ligand-receptor complexes will be studied to understand the molecular determinants for trafficking of membrane components. The role in endocytic trafficking of specific proteins such as cytoskeleton-associated motors or small GTP-binding proteins will be tested in living cells by microinjection of antibodies, transfection with normal and altered proteins, and analysis of mutant cell lines. It is expected that these studies will lead to a fairly complete map of the endocytic pathway in nonpolarized cells within five years. These studies should help specialists in the field of endocytosis by providing an improved basis for understanding and interpreting in vitro experiments. They should also aid nonspecialists who are interested in determining how endocytosis might be involved in a variety of biological processes.

DESCRIPTION [Verbatim from application]: The overall goal of this study is to understand how leukocytes respond to external stimuli and migrate to sites of infection and inflammation. Previous efforts have been focused on understanding the role of changes in intracellular free calcium [Ca2+i], in regulating adhesive interactions and the cytoskeleton. It was found that transient increases in [Ca2+i] are required for neutrophils to dissociate from vitronectin and fibronectin. The [Ca2+i]-sensitive binding to these matrix proteins is via alpha v beta 3 and alpha 5 beta 1 integrins, respectively. Under normal conditions, it was found that both of these integrins are distributed on the adherant membrane with a gradient that is higher at the front of the cell. These integrins are also in endocytic vesicles. When [Ca2+i] transients are blocked, the integrins are found at the rear of the cells on the adherant membrane, and endocytic vesicles do not contain the integrins. Based on these and other data it was proposed that [Ca2+i] transients are required to release the alpha v beta 3 and alpha 5 beta 1 integrins from tight binding and that after release they are internalized and recycled toward the front of a migrating cell. One aim of the proposed research is to investigate the oriented recycling in migrating neutrophils. Digital fluorescence microscopy, confocal microscopy, and electron microscopy will be used to examine the endocytic recycling pathways in neutrophils, and the passage of integrins through these pathways will be examined in detail. The role of microtubule-based vesicle motors will be examined by disruption of dynein motor function in neutrophils and neutrophil-like HL-60 cells by overexpression and/or cytoplasmic delivery of p50-dynamitin. Myosin II is a major cytoskeletal protein that is activated by increases in [Ca2+i]. The role of myosin II in neutrophil migration on 2D substrates and through natural 3D matrices will be examined. Using antibodies to myosin II and affinity purified antibodies to the phosphorylated form of myosin light chain, the distribution of myosin II and its activation under various conditions will be determined by immunofluorescence. Myosin II function will be inhibited by delivery of inhibitory peptides to the cytoplasm of migrating cells. Finally, the role of myosin and of oriented recycling will be examined in neutrophils migrating through endothelial cell monolayers and through natural biological matrices, which resemble the physiological sites of neutrophil function.

This grant will partially support travel, registration and subsistence for participants in the 16th Gordon Conference on Lysosomes to be held July 3- 8, 1994 at the Proctor Academy, Andover, NH. The conference will provide an opportunity for formal presentations and informal discussions on a variety of topics related to membrane traffic and intracellular organelles in eukaryotic cells. The meeting will cover basic aspects of cell biology as well as applications to specific disease processes for which intracellular membrane traffic is important for understanding pathophysiology and for designing therapies. Session topics will include: Formation of coated membranes and vesicles, Mechanisms of endocytic sorting, Molecular control of vesicle targeting and fusion, Membrane traffic in polarized cells and neurons, Processing of proteins in pathological processes, Lipids and GPI-anchored proteins, and Intracellular pathogens. These sessions will provide up to date information of direct relevance to a variety of disease-related topics, including diabetes, heart disease, neuronal degeneration, intracellular parasites, lysosomal storage disorders, vaccine development, and others. The speakers in the regular program will include several recognized world leaders in this field as well as younger investigators. All participants will be encouraged to present posters, and a few abstracts from young investigators will be selected in advance to be included for short lectures. The goal of the Conference is to provide a forum for the free exchange of new findings that will lead to new ideas and collaborations among scientists from a variety of backgrounds.

digital imaging; confocal scanning microscopy; biomedical equipment purchase; biomedical facility;

DESCRIPTION (Abstract): Microglia are immune-system cells associated with senile plaques containing B-amyloid (AB) in Alzheimer's disease (AD). Although microglia are an integral part of senile plaques, their role in the development of A is not known. Because microglia are phagocytic cells, it has been suggested that microglia may function as plaque-attacking scavenger cells. It has also been suggested that microglia may participate in the development of Alzheimer's disease by initiating an inflammatory response. Microglia bind and internalize fibrillar microaggregates of AB that resemble those present in dense AD plaques. The internalized fibrillar AB is delivered to late endosomes and lysosomes, but it is only partially degraded. Undigested fibrillar AB is released over a period of several days. Soluble AB adsorbed to carrier proteins is also degraded poorly by microglia. In contrast to microglia, cultured macrophages can degrade fibrillar AB rapidly and completely. We will compare the two cell types to determine why macrophages are more effective at degradation of AB, and we will use this information to boost the degradative capability of microglia in culture. We will test the hypothesis that microglia can participate in the accumulation of amyloid plaques by impaired degradation, by promotion of plaque formation in acidic endosomes, by remodeling of plaque in endosomes and lysosomes, and by release of amyloid-like particles. We will study the interaction of plaque with microglia using digital fluorescence microscopy, confocal microscopy, electron microscopy, and biochemical analysis. We will analyze the mechanisms of degradation of AB amyloid in microglia and also determine whether soluble AB peptides become incorporated into insoluble AB in the endosomes and lysosomes of microglia.

DESCRIPTION (provided by applicant): The overall goal of this project is to understand how molecules are sorted to maintain the distinct protein and lipid compositions of organelles. The focus is on endocytic sorting, but endocytic pathways intersect with vesicular and non-vesicular transport pathways involving other organelles, including the ER and the Golgi. Methods have been developed to characterize endocytic pathways in cells morphologically and kinetically, including measurements of kinetics and efficiency of many sorting steps. Several proteins have been shown to play some role in sorting, but in many cases the precise role remains uncertain. The proposed work will test specific roles in well-defined trafficking steps for several key sorting proteins. The methods used will include mutation of binding sequences in cargo proteins, knockdown of sorting proteins by RNAi, and rapid inactivation by microinjection of inhibitors, chemically-induced crosslinking, or photophysical inactivation. Effects on trafficking will be measured by well-characterized quantitative optical microscopy assays. Cargo proteins to be studied include transferrin receptors, the cation-independent mannose-6-phosphate receptor, furin, and TGN38. The sorting proteins to be examined include clathrin, adaptins, GGAs, and the retromer complex. Protein traffic is intimately linked with lipid traffic and cholesterol levels. Novel methods have been developed to directly observe sterol trafficking in living cells, and these will be used to analyze the mechanisms for vesicular and non-vesicular sterol traffic. The role of the StAR family of proteins in nonvesicular sterol transport will be evaluated. The effects of changes in expression levels of various ABC transporters and stearoyl-coA-desaturase on the distribution and trafficking of sterol in cells will be measured. Changes in the biophysical properties of cell membranes under these conditions will be analyzed by fluorescence and by ESR of spin-labeled lipids. A novel mechanism by which unsaturated fatty acids alter the trafficking of the sterol response element binding protein will be characterized.

Early in the pathogenesis of atherosclerosis, aggregated, oxidized lipoprotein particles become attached to proteins in the subendothelial matrix of the affected arteries. Monocytes (Mos) that traverse the overlying endothelium come into contact with these matrix-bound lipoprotein particles, engulf them, and eventually take up large amounts of lipid, developing into lipid-engorged macrophage foam cells, which promote further growth of the atherosclerotic plaque. We hypothesize that alterations in the plasma membrane cholesterol levels in Mos and macrophages caused by interactions with these matrix-bound lipoproteins may be partially responsible for lesion growth. Based on our preliminary studies, we postulate that retained and aggregated lipoproteins alter actin dynamics in Mos and macrophages in the subendothelial space, and therefore inhibit the migration and phagocytic function of these cells. This may promote the retention of cells in the tissue and thus the growth of atherosclerotic plaques. The specific aims of this grant are designed to test this hypothesis. Numerous studies have investigated the effects of reducing membrane cholesterol levels on cellular functions, but far fewer have looked at the effects of the potentially more physiologically significant event of increasing membrane cholesterol levels. Our preliminary data indicate that overloading a Mo's plasma membrane with cholesterol decreases its migration rate in a three dimensional (3D) collagen gel. Similarly, our studies with neutrophils showed that cholesterol depletion inhibited Rac GTPase-mediated actin reorganization and migration. We propose to first quantify the extent to which stimulated actin reorganization in Mos and macrophages is sensitive to raising or lowering membrane cholesterol levels, and then we will study how these changes affect the actin-dependent functions of migration and phagocytosis. Next, we will directly test the idea that effects on migration and phagocytic function can be attributed to disruption of activation and/or targeting of Rho GTPases (i.e. Rho, Rac, and Cdc42). Initially we will use pharmacological means to alter membrane cholesterol, and later we will use progressively more physiological cholesterol modulating scenarios from isolated lipoproteins to matrix-embedded lipoproteins that mimic an atherogenic environment.

Early in the pathogenesis of atherosclerosis, aggregated, oxidized lipoprotein particles become attached to proteins in the subendothelial matrix of the affected arteries. Monocytes (Mos) that traverse the overlying endothelium come into contact with these matrix-bound lipoprotein particles, engulf them, and eventually take up large amounts of lipid, developing into lipid-engorged macrophage foam cells, which promote further growth of the atherosclerotic plaque. We hypothesize that alterations in the plasma membrane cholesterol levels in Mos and macrophages caused by interactions with these matrix-bound lipoproteins may be partially responsible for lesion growth. Based on our preliminary studies, we postulate that retained and aggregated lipoproteins alter actin dynamics in Mos and macrophages in the subendothelial space, and therefore inhibit the migration and phagocytic function of these cells. This may promote the retention of cells in the tissue and thus the growth of atherosclerotic plaques. The specific aims of this grant are designed to test this hypothesis. Numerous studies have investigated the effects of reducing membrane cholesterol levels on cellular functions, but far fewer have looked at the effects of the potentially more physiologically significant event of increasing membrane cholesterol levels. Our preliminary data indicate that overloading a Mo's plasma membrane with cholesterol decreases its migration rate in a three dimensional (3D) collagen gel. Similarly, our studies with neutrophils showed that cholesterol depletion inhibited Rac GTPase-mediated actin reorganization and migration. We propose to first quantify the extent to which stimulated actin reorganization in Mos and macrophages is sensitive to raising or lowering membrane cholesterol levels, and then we will study how these changes affect the actin-dependent functions of migration and phagocytosis. Next, we will directly test the idea that effects on migration and phagocytic function can be attributed to disruption of activation and/or targeting of Rho GTPases (i.e. Rho, Rac, and Cdc42). Initially we will use pharmacological means to alter membrane cholesterol, and later we will use progressively more physiological cholesterol modulating scenarios from isolated lipoproteins to matrix-embedded lipoproteins that mimic an atherogenic environment.

DESCRIPTION (provided by applicant): Atherosclerosis results in part from the accumulation of lipoproteins in the arterial wall, recruitment of monocytes/macrophages to the region, and formation of lipid-laden macrophages, known as foam cells. Foam cell formation is dependent on interactions with lipoproteins and subsequent cellular cholesterol transport among specific intracellular compartments. This project will examine how characteristics of both the lipoprotein and the macrophage lead to foam cell formation and the pathogenesis of atherosclerosis. Several key aspects of this process will be examined. First, many of the lipoproteins in the wall of blood vessels become aggregated and tightly bound to extracellular matrix components, and this prevents their internalization by simple endocytic processes. When macrophages encounter these aggregated lipoproteins, there is a prolonged period of contact before the lipoprotein is internalized. Preliminary data indicate that macrophages form an unusual extracellular, acidic, digestive compartment during this contact period. The acidification of this extracellular compartment will be characterized further using quantitative fluorescence microscopy, and the mechanism for acidification will be examined. The structure of the extracellular compartment will be examined using intermediate voltage electron microscopy and tomographic imaging. The mechanism for delivering acid hydrolases into the compartment will also be examined, including the role of calcium signaling and specific SNARE molecules in mediating lysosomal fusion with the plasma membrane. The presence of lysosomal acid lipase in this compartment would lead to hydrolysis of cholesterol esters and release of free sterol, which can be inserted locally in to the plasma membrane. Well- characterized fluorescent sterols and their ester derivatives will be incorporated into lipoproteins to monitor this process directly. Other catabolic processes (e.g., protein degradation) in this compartment will be investigated. Delivery of cholesterol to the plasma membrane leads to activation of signal transduction mechanisms, including the small GTPase, Rac, leading to increased actin polymerization. The consequences of sterol delivery from aggregated lipoproteins to macrophages on Rac, Rho and PI3-kinase activity and actin assembly will be examined. The possibility that increased actin assembly enlarges the extracellular, acidic, digestive compartment will be examined using inhibitors of these signaling proteins. Different classes of macrophages may respond differently to aggregated lipoproteins. The interactions of macrophages derived from classical and nonclassical monocytes with aggregated lipoproteins will be compared in tissue culture. The abundance of macrophages and foam cells derived from classical and nonclassical Mo subsets in atherosclerotic lesions in mouse models of atherosclerosis will also be examined. PUBLIC HEALTH RELEVANCE: This project examines the early steps leading to an atherosclerotic lesion as a consequence of the interaction between an immune system cell, the macrophage, with lipoproteins in the wall of blood vessels. These atherosclerotic lesions can lead to heart disease and stroke, which are major causes of disability and death of Americans. Research findings based on these studies may lead to new treatments to prevent or reverse the formation of atherosclerotic lesions.

DESCRIPTION (provided by applicant): Although multiple lines of evidence link beta-amyloid peptides to the pathogenesis of Alzheimer's disease, the molecular mechanism whereby beta-amyloid is involved remains unknown. Synaptic dysfunction is an early event in Alzheimer's disease and increasing evidence indicates that the aberrant accumulation of beta- amyloid within neurons is critical for synaptic dysfunction. Specifically, a triple transgenic mouse was described in which physiological alterations implicated in memory were altered with the onset of intraneuronal beta-amyloid accumulation and prior to plaques and tangles. Employing immuno-gold electron microscopy, we reported in transgenic mutant APR mice that develop age-related beta-amyloidosis the accumulation of beta-amyloid especially in late endosomal vesicles of distal processes and synaptic compartments, which at times were associated with subcellular morphological alterations consistent with degeneration. In a subsequent study, we demonstrated that beta-amyloid oligomerization begins within processes and synaptic compartments and is consistently linked with neurodegeneration. Moreover, we found by Western blot, immunofluorescence microscopy and immuno-electron microscopy that neurons from amyloid precursor protein (APR) mutant transgenic mice with time in culture paralleled the subcellular beta- amyloid accumulation and Alzheimer's disease-like synaptic alterations observed in brain in vivo. We hypothesize that intraneuronal beta-amyloid accumulation induces synaptic dysfunction by impairing multivesicular body sorting and the ubiquitin proteasome system in neurons. We propose studies in mutant APR transgenic neurons in culture to elucidate the biological mechanism leading to synaptic dysfunction. Our results indicate that mutant APR transgenic neurons have alterations in endocytosis, differential pre- and post-synaptic proteins and the ubiquitin proteasome system. A better understanding of the mechanism whereby beta-amyloid is involved in synaptic dysfunction and Alzheimer's disease pathogenesis may be important for devising more effective treatments for Alzheimer's disease.

DESCRIPTION (provided by applicant): This application seeks funds to purchase a JEOL JEM 1400 electron microscope to serve as the sole EM available for research in a Core Facility at Weill Cornell Medical College and the Hospital for Special Surgery. The Analytical Microscopy Laboratory (AML), located at the Hospital for Special Surgery, has agreed to join with Electron Microscopy &Histology Core Facility at Weill Cornell Medical College (WCMC) to share the costs of operating and maintaining the transmission electron microscopy services for both institutions. The two institutions are immediately adjacent to each other and share several core facilities. Sharing the use of a modern electron microscope will enhance the research capabilities at both institutions, and it will provide increased access to expert technical assistance. The participants in this application have projects that address topics with applications to translational medicine including: receptor trafficking within cells;neural development and plasticity;angiogenesis;cell differentiation, and musculoskeletal metabolism and repair. The success of this joint venture is dependent on the quality of the equipment. The requested microscope, the JEOL JEM 1400 is a state of the art 120 kV electron microscope with added advantage that it is possible to upgrade the instrument to include advanced feature modules such as tomography and cryo in the field for relatively modest costs. This will enable the joint facilities to continue to meet the needs of the research faculties at both WCMC and HSS in future years without needing to invest in another large full-scale instrument.

DESCRIPTION (provided by applicant): This proposal seeks funds to purchase an Olympus FV1000MPE multiphoton microscope to be used as the main instrument in a core facility at the Weill Cornell Medical College. The core facility serves translational and basic medical science investigators. In addition to investigators at Weill Cornell, researchers from the Hospital for Special Surgery will be users of the facility. Additionally, this instrument will serve as the major instrument for a large collaborative project with scientists at Cornell University in Ithaca who are developing novel methods, including multiphoton endoscopy, for use in human patient care. This collaborative project is also a component of the Clinical and Translational Science Center Project based at Weill Cornell. The largest user of the facility will be a collaborative group of surgeons, pathologists, microscopists, engineers, and physicists who are examining unstained fresh human biopsy samples to create an atlas of normal and diseased human tissue as seen by autofluorescence and second harmonic generation. Preliminary data suggest that such images can provide diagnostic information comparable to conventional histopathology for certain types of cancers. SHG from collagen will also be used for examining human bone biopsy specimens for studies of osteoporosis. Several studies will carry out imaging in living animals, and several of these projects will benefit from synergistic interactions based on the use of the FV1000MPE. For example, three investigators will examine events in the brain, and two of these collaborate on studies of the formation and degradation of Alzheimer's disease amyloid plaques. Two projects will examine macrophages and dendritic cells in mice and will investigate properties such as cell migration. The FV1000MPE is very well suited to serve the needs of this group of investigators. It has superb optics that are optimized for multiphoton microscopy and for relatively effective imaging deep into tissue. It is easy to use, which will allow rapid training of investigators with diverse backgrounds. The microprobe objectives are very advantageous for imaging in animals because of their small diameter, and these will also be very useful for examination of irregularly shaped surgical specimens, which cannot be cut or altered before being sent for pathology processing.

? DESCRIPTION (provided by applicant): Niemann Pick C disease is a rare, neurodegenerative, lipid storage disorder. Approximately 95% of the disease is caused by mutations in NPC1, a late endosomal membrane protein that functions in export of lipoprotein-derived cholesterol. The most prevalent NPC1 mutation, I1061T, produces a protein that is misfolded and rapidly degraded. Histone deacetylase inhibitors (HDACi) recently have been shown to reduce the accumulation of cholesterol and other lipids found in patient cells harboring the NPC1I1061T and other mutations. This beneficial effect is associated with decreased endoplasmic reticulum-associated degradation and enhanced delivery of the mutant NPC1 proteins to late endosomes and lysosomes. With the recent generation in our laboratory of a humanized mouse model in which the I1061T mutation knocked into the murine NPC1 locus, it is possible to examine the effect of HDACi on NPC1 stability in vivo. We hypothesize that treatment with an HDACi in the NPC1I1061T knockin model of NPC1 disease will increase levels of the mutant NPC1I1061T protein, slowing progression of neurodegeneration and prolonging survival. The therapeutic potential of HDACi for treatment of NPC1 disease is being explored in through a collaboration involving pharmaceutical partners and an HDACi collaborative involving investigators from NIH (NICHD/NCATS), Weill Cornell Medical College, University of Notre Dame, Albert Einstein College of Medicine, and Washington University, along with the Ara Parseghian Medical Research Foundation. The goals of this proposal are to identify orally-available, CNS-penetrant HDAC-selective compounds using cell-based screens; to evaluate in vivo in the NPC1I1061T knockin model candidate HDACi compounds; and to develop effective therapeutic regimens for testing of the HDACi in clinical trials. The proposed in vivo studies further will provide valuable data for initial dosing protocols and biomarker monitoring in future human trials.

Cholesterol plays an essential role in determining the properties of biological membranes. Control of cholesterol levels in organelles depends on sterol transport mechanisms that are poorly understood. Novel methods to study sterol transport among organelles quantitatively have been developed, and this is leading to precise models for transport among organelles. Preliminary data show that a widely expressed, sterol- regulated, soluble sterol transporter, STARD4, plays an important role in nonvesicular transport of cholesterol, and its role and mechanism of transport will be studied. STARD4 is an important component of a complex sterol regulatory network. Recent work showed that STARD4 is responsible for about 25% of sterol transport between two major pools ? the plasma membrane and the endocytic recycling compartment - in U2OS cells. The role of STARD4 will also be assessed in macrophages, a cell type in which cholesterol metabolism plays a major role in development of atherosclerosis, and in HepG2 cells, a model for hepatocytes. In preliminary studies it has been found that STARD4 has unique interactions with phosphatidylinositol phosphates (PIPs). STARD4 extraction of sterol from membranes is accelerated nearly 10-fold when the membranes contain PI(4,5)P2, but there is no effect of PI(4,5)P2 in membranes accepting sterol from STARD4. Conversely, PI(3,5)P2 or PI5P in acceptor membranes accelerates transfer ?10-fold with no effect when these PIPs are in donor membranes. PI3P has a similar but smaller effect. The role of STARD4 in sterol transport to late endosomes, lipid droplets, and autophagosomes (i.e., organelles with good acceptor PIPs) will be analyzed. Molecular dynamics simulations of interactions of STARD4 with membranes is identifying potential sites of interaction with PIPs. The effects of mutations of these sites will be assayed in a liposome to liposome sterol transport assay. Mutant STARD4 molecules with altered PIP sensitivity will be expressed in cells to determine effects on sterol transport and distribution. In preliminary studies, mutants identified by molecular dynamics were confirmed by tests in the liposome assay, and it was found that they do affect transport of sterol among organelles. X-ray crystallography has identified differences between human STARD4 with and without bound sterol. NMR spectroscopy is being used to analyze STARD4 protein dynamics and to determine the sites of interaction with lipids containing various PIPs.